Phenylalanine ammonia lyases (PAL, EC 4.3.1.5) catalyze the deamination of phenylalanine to trans-cinnamic acid and ammonia (FIG. 5). In nature, they facilitate the committed step in phenylpropanoid pathways to produce lignins, coumarins, and fiavonoids. Depending on the source of the enzyme, PALs may show varying selectivity towards phenylalanine and tyrosine derivatives (those active on tyrosine derivatives are known as tyrosine ammonia lyases (TALs)). Histidine ammonia lyases (HALs, EC 4.3.1.3) are distinct from PALs in that they have a substrate preference for histidine over phenylalanine or tyrosine. HALs catalyze the abstraction of ammonia from histidine to form urocanoic acid.
Most of the phenylalanine ammonia lyases (PALs) currently described are from plant origins where the enzyme plays a central role in plant metabolism. Recently, PALs have been identified in fungi and a very limited number have been identified in bacteria. HALs have also been identified in plants and fungi. Unlike PALs, HALs have been found to be widespread in bacteria. Synthetic applications of HALs tend to be rather limited compared to PALs. Some niche applications have been developed such as the synthesis of radiolabeled urocanoic acids as tracers of histidine metabolism. There may be potential to expand applications of HALs by discovery of enzymes with greater stability to oxygen.
Up until the late 1990s, it was thought that histidine and phenylalanine ammonia lyases utilized a dehydroalanine cofactor in their catalytic mechanism. However X-ray crystallographic studies have shown that the cofactor is actually 3,5-dihydro-5-methylidine-4H-imidazol-4-one (MIO), which is formed by cyclization and dehydration of a conserved active site Ala-Ser-Gly sequence. Enzyme mechanistic studies have led to two main proposals on the catalytic mechanism of phenylalanine ammonia lyases (PALs), as shown in FIGS. 6a and 6b. In both mechanisms A and B, the MIO group acts as a powerful electrophile; in mechanism A the MIO group reacts with the amino group of Phe, while in mechanism B it reacts with the aromatic side chain in a Friedel-Crafts-type reaction.
Applications of PALs include the manufacture of phenylalanine and tyrosine as well as phenylalanine and tyrosine derivatives. Applications include utilizing the enzymes to degrade phenylalanine, tyrosine, and derivatives to manufacture cinnamic acid, para-hydroxycinnamic acid and derivatives. Fields of use include manufacture of bulk and fine chemicals for industrial, medicinal and agricultural use, as well as the direct application of the enzymes themselves for an enzyme substitution therapy.
For example, PALs have been investigated for an enzyme substitution therapy for the treatment of phenylketonuria (PKU), an inherited metabolic disease caused by a deficiency of the enzyme phenylalanine hydroxylase. PKU is one of the most commonly inherited metabolic disorders, affecting an estimated 50,000 people in the developed world or 30,000 people in the United States. It occurs in approximately 1 in 10,000 (0.01%) babies born in the US. PKU is an inborn error of amino acid metabolism caused by a phenylalanine hydroxylase defect (PAH). Untreated patients with PKU often show mental retardation or otherwise impaired cognitive function. Currently the only treatment for PKU is strict dietary control via a low-phenylalanine diet. A few pharmaceutical modalities to treat PKU are under investigation. One of these approaches is the use of phenylalanine ammonia-lyase (PAL) as an enzyme replacement therapy. Several reports of applying a PAL (R. toruloides) to decrease phenylalanine serum levels in murine models have been published. However, developing a form of this enzyme with sufficiently high activity and stability has proven difficult. One concept was the application of PAL as an oral treatment to break down phenylalanine in the gut. PAL therapy is also being considered for use with CLEC™ (crystallized enzyme crystal) methodology to stabilize the enzyme for oral delivery. Degradation of phenylalanine by PAL treatment yields trans-cinnamate which has very low toxicity. In addition, PAL therapy has the advantage that it does not require exogenous cofactors to degrade Phe. There is a need for more PAL enzymes to extend the utility of this versatile enzyme class, especially PALs of bacterial origin. Bacterial PALs potentially offer greater catalytic versatility than plant and fungal enzymes since their natural cellular roles are likely more diverse.